Unusually high number of HLA-C recognition in CD8+ T cells
Responses could not be validated through TCR-specific confirmation assays
The Killer Immunoglobin Receptor (KIR) leads to false positive responses
Figure 1: KIR binding and blocking (Source: Biorender)
Figure 2: Flowchart of the project
Data:
High-throughput experiment
Thousands of SarsCov2 peptides binding to T-cells
TCR-dependent or TCR-independent (i.e.: KIR-mediated)?
3 cohorts:
AP: Leukemia patients who have had COVID-19 (n = 12)
BS: Healthy nurses who have had COVID-19 (n = 17)
BC: Healthy donors (n = 15)
Methods:
DNA-barcoding: Tagging each peptide-HLA pair uniquely
Flow cytometry: Sorting T-cells based on TCR vs KIR binding
| AP (N=13465) |
BS (N=7840) |
BC (N=10491) |
Overall (N=31796) |
|
|---|---|---|---|---|
| KIR_blocking | ||||
| 1 | 7541 (56.0%) | 4019 (51.3%) | 7877 (75.1%) | 19437 (61.1%) |
| 0 | 5924 (44.0%) | 3821 (48.7%) | 2614 (24.9%) | 12359 (38.9%) |
| log_fold_change | ||||
| Mean (SD) | -1.15 (3.29) | -2.25 (3.95) | -1.49 (3.69) | -1.53 (3.62) |
| Median [Min, Max] | -0.182 [-13.4, 9.71] | -0.508 [-12.0, 8.64] | -0.313 [-12.4, 11.2] | -0.290 [-13.4, 11.2] |
| protein | ||||
| ORF1 | 7239 (53.8%) | 4447 (56.7%) | 6103 (58.2%) | 17789 (55.9%) |
| Nucleocapsid | 707 (5.3%) | 434 (5.5%) | 548 (5.2%) | 1689 (5.3%) |
| Spike | 2619 (19.5%) | 1581 (20.2%) | 2062 (19.7%) | 6262 (19.7%) |
| ORF6 | 68 (0.5%) | 42 (0.5%) | 65 (0.6%) | 175 (0.6%) |
| Membrane | 785 (5.8%) | 465 (5.9%) | 545 (5.2%) | 1795 (5.6%) |
| ORF3 | 540 (4.0%) | 345 (4.4%) | 467 (4.5%) | 1352 (4.3%) |
| ORF7 | 288 (2.1%) | 175 (2.2%) | 227 (2.2%) | 690 (2.2%) |
| Envelope | 119 (0.9%) | 73 (0.9%) | 91 (0.9%) | 283 (0.9%) |
| ORF10 | 119 (0.9%) | 73 (0.9%) | 91 (0.9%) | 283 (0.9%) |
| ORF8 | 322 (2.4%) | 205 (2.6%) | 292 (2.8%) | 819 (2.6%) |
| Unknown | 659 (4.9%) | 0 (0%) | 0 (0%) | 659 (2.1%) |
| HLA | ||||
| HLA-C04:01 | 300 (2.2%) | 600 (7.7%) | 2400 (22.9%) | 3300 (10.4%) |
| HLA-C06:02 | 2695 (20.0%) | 1086 (13.9%) | 1164 (11.1%) | 4945 (15.6%) |
| HLA-C07:01 | 4014 (29.8%) | 2950 (37.6%) | 2655 (25.3%) | 9619 (30.3%) |
| HLA-C07:02 | 6456 (47.9%) | 3204 (40.9%) | 4272 (40.7%) | 13932 (43.8%) |
Table 1: Barracoda data
~ 32 000 bar-codes (patient-HLA-peptide combinations)
44 patients
4 HLA types
11 protein types
The number of observations reduced drastically during wrangling:
Original: 31796
w. Flowjo: 22819
Significant: 542
Paired data to compare TCR-dependent and TCR-independent responses:
This further reduced the dataset size:
Original: 31796
Significant: 542
Paired: 22
11 pairs
1 normal
1 KIR-blocked
\[ Est. Freq = \left( \frac{count_{barcode}}{count_{total}} \cdot 100 \% \right) \cdot PE \]
The fraction of T cells recognising the specified peptide
C0602 is a true response
AP cohort may have lower KIR expression due to pathology
BC cohort show significant response for C0401
False positives are removed in BS cohort for C0602
C0701 and C0702 show inconsistent responses
The paired dataset was really small, hard to draw reliable conclusions
New info from this research group indicates that the KIR-antibody used in this experiment only blocked KIR-C1 but not -C2
C0401 and C0601 are C2 ligands and were therefore not blocked
This would explain the unreliable results, and variation between cohorts
R4BDS - Group 25